Search

Background Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. Methods MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. Results The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. Conclusions Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.

Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB-induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild-type HSP27-overexpressing cells and the phospho-mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPK, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPK, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27.

Resveratrol, a polyphenolic compound that is present in grape skins, berries and red wine, may be beneficial for human health through its anti‑inflammatory and anti‑oxidant effects. It has been previously demonstrated that resveratrol exerts its biological effects primarily via sirtuin 1 (SIRT1) activation. We previously reported that triiodothyronine (T3) induces osteocalcin synthesis in osteoblast‑like MC3T3‑E1 cells, and that p38 mitogen‑activated protein (MAP) kinase mediates the T3‑stimulated synthesis of osteocalcin. The present study investigated the effect of resveratrol on T3‑induced osteocalcin synthesis and its underlying mechanism in MC3T3‑E1 cells. Cultured cells were stimulated with T3, and osteocalcin release from MC3T3‑E1 cells was measured by ELISA and phosphorylation of p38 MAP kinase was analyzed by western blotting. Resveratrol significantly suppressed the release of osteocalcin stimulated by T3, and SRT1720, a SIRT1 activator, significantly reduced T3‑induced osteocalcin release. The expression level of osteocalcin mRNA stimulated by T3 was significantly attenuated by resveratrol and T3‑induced transactivation activity of the thyroid hormone‑responsive element was significantly diminished by resveratrol. However, only limited effects of resveratrol on the T3‑induced phosphorylation of p38 MAP kinase were observed. The results of the present study demonstrated that resveratrol suppresses T3‑stimulated osteocalcin synthesis at a point upstream of transcription in osteoblasts, and that the inhibitory effect of resveratrol is mediated, at least partially, through SIRT1 activation. These results indicate that there may be a novel role for the polyphenol in the modulation of bone metabolism.

Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia‑inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis‑osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2α‑induced osteoprotegerin (OPG) synthesis without affecting interleukin‑6 (IL‑6) production in osteoblast‑like MC3T3‑E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogen‑activated protein (MAP) kinase and stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL‑6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1‑stimulated synthesis of OPG and IL‑6 were investigated in osteoblast‑like MC3T3‑E1 cells. The concentrations of OPG and IL‑6 were measured using relevant ELISA kits. OPG mRNA was measured by semi‑quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1‑induced release of OPG and OPG mRNA expression levels without affecting the release of IL‑6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1‑induced OPG release and OPG mRNA expression levels; however, it had little effect on IL‑6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1‑stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1‑stimulated OPG synthesis without affecting IL‑6 production in osteoblasts.

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII.

AMP-activated protein kinase (AMPK) is firmly established as a central regulator of cellular energy homeostasis. We have previously reported that prostaglandin E1 (PGE1) stimulates the synthesis of osteoprotegerin through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. The present study investigated the involvement of AMPK in PGE1-induced osteoprotegerin synthesis in MC3T3-E1 cells. The levels of osteoprotegerin were measured using an enzyme-linked immunosorbent assay, while the phosphorylation of AMPK, acetyl-CoA carboxylase, p38 MAP kinase and SAPK/JNK were analyzed by western blotting. In addition, the mRNA expression levels of osteoprotegerin were determined by a reverse transcription-quantitative polymerase chain reaction. It was revealed that PGE1 significantly induced the phosphorylation of the α and β subunits of AMPK in a time-dependent manner (P

Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F2α (PGF2α) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF2α induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF2α-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1α. Both mimosine and deferoxamine significantly suppressed the PGF2α-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF2α-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF2α was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF2α‑induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.

Polyphenolic compounds in foods and beverages have beneficial effects on human health. (-)-Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), a major flavonoid in green tea and a major phenolic acid in coffee, respectively, have potent properties, including antioxidative effects. Our previous study demonstrated that p70 S6 kinase acts as a negative regulator in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, the effects of EGCG and CGA on the TNF-α-stimulated interleukin‑6 synthesis were investigated in MC3T3‑E1 cells. EGCG and CGA significantly enhanced TNF-α-stimulated interleukin-6 release. In addition, the interleukin-6 mRNA expression levels induced by TNF‑α were supported by EGCG, as well as CGA. EGCG markedly attenuated the TNF-α-induced phosphorylation of p70 S6 kinase whereas CGA failed to affect the phosphorylation. These results strongly suggest that EGCG and CGA enhance the TNF-α-stimulated interleukin-6 synthesis in osteoblasts, and that the amplifying effect of EGCG, but not CGA, is exerted via inhibiting p70 S6 kinase.

Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.

Resveratrol is a naturally occurring polyphenol that possesses health‑related properties, and is predominantly found in grapes and berries. Bone morphogenetic protein‑4 (BMP‑4) stimulates osteocalcin synthesis via p38 mitogen‑activated protein (MAP) kinase in osteoblast‑like MC3T3‑E1 cells. The present study aimed to investigate the effects of resveratrol on BMP‑4‑induced osteoprotegerin (OPG) synthesis in MC3T3‑E1 cells. Resveratrol alone had no effect on OPG expression levels, but significantly enhanced BMP‑4‑induced OPG release. In addition, resveratrol markedly amplified the mRNA expression levels of BMP‑4‑induced OPG. SB203580 is an inhibitor of p38 MAP kinase, which was shown to suppress BMP‑4‑stimulated OPG release. BMP‑4‑induced phosphorylation of p38 MAP kinase was also enhanced by resveratrol. Furthermore, SB203580 significantly reduced the resveratrol‑induced amplification of BMP‑4‑stimulated OPG release. These results suggested that resveratrol was able to upregulate BMP‑4‑stimulated OPG synthesis via the amplification of p38 MAP kinase activity in osteoblasts.

We previously showed that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rac, which is a member of the Rho family of small GTPases, in the TGF-β-stimulated VEGF synthesis in MC3T3-E1 cells. TGF-β markedly increased the levels of GTP-bound Rac. NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, significantly increased both the release of VEGF and the mRNA expression levels induced by TGF-β. In addition, the release of VEGF stimulated by TGF-β was amplified in Rac-knock down cells. Meanwhile, SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the TGF-β-stimulated VEGF release. However, the phosphorylation of Smad2 or Smad3 induced by TGF-β was hardly affected by NSC23766. On the other hand, NSC23766 enhanced the TGF-β-induced phosphorylation of p38 MAP kinase without affecting the phosphorylation of p44/p42 MAP kinase or SAPK/JNK. Furthermore, the phosphorylation of p38 MAP kinase induced by TGF-β was markedly upregulated in the Rac-knock down cells. These results strongly suggest that Rac negatively regulates the TGF-β-stimulated VEGF synthesis via the inhibition of p38 MAP kinase in osteoblasts.

Resveratrol, which is found in grape and berry skins and red wine, is generally known to be beneficial for human health due to its anti-inflammation and antioxidant effects. We have recently reported that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis through Smad-independent pathways, such as the p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways, in osteoblast-like MC3T3-E1 cells. The aim of the present study was to investigate the effect of resveratrol on the TGF-β-induced VEGF synthesis and the mechanism in osteoblast-like MC3T3-E1 cells. Resveratrol significantly suppressed the TGF-β-stimulated release of VEGF and the VEGF mRNA expression levels. SRT1720, a synthetic sirtuin 1 (SIRT1) activator, also reduced the VEGF release and the mRNA levels. With regard to the intracellular signaling in the TGF-β-stimulated VEGF synthesis, resveratrol and SRT1720 significantly attenuated the phosphorylation of p44/p42 MAP kinase and SAPK/JNK stimulated by TGF-β; however, the TGF-β-induced phosphorylation of Smad2 and p38 MAP kinase was hardly affected by resveratrol or SRT1720. These results strongly suggest that the TGF-β-stimulated VEGF synthesis is suppressed by resveratrol through the inhibition of p44/p42 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is mediated, at least in part, via SIRT1 activation.

We have previously demonstrated that ristocetin, an activator of GPIb/IX/V, induces the release of soluble CD40 ligand (sCD40L) via thromboxane A2 production in human platelets. It has been shown that thromboxane A2 induces the activation of Rho-kinase, a downstream effector of Rho, in human platelets. In the present study, we investigated the exact roles of Rho-kinase in thromboxane A2-induced platelet activation. We found that U46619, a thromboxane receptor (TP) agonist, induced the phosphorylation of cofilin, a target of Rho-kinase signaling, and that the cofilin phosphorylation by U46619 was suppressed by Y27632 or fasudil, specific inhibitors of Rho-kinase. Y27632 and fasudil markedly decreased large platelet aggregate formation by U46619. The release of sCD40L and secretion of platelet-derived growth factor (PDGF)-AB stimulated by U46619 were inhibited by Y27632 and fasudil. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, reduced the sCD40L release and PDGF-AB secretion. Y27632 and fasudil failed to affect the phosphorylation of p38 MAP kinase whereas SB203580 had little effect on the phosphorylation of cofilin induced by U46619. In conclusion, our results strongly suggest that Rho-kinase regulates thromboxane A2-induced human platelet activation independently of p38 MAP kinase.

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.

Heat shock protein 90 (HSP90) is an ATP‑dependent ubiquitous molecular chaperon which is important in cell homeostasis. The authors previously demonstrated that bone morphogenetic protein (BMP)‑4 stimulates osteoprotegerin (OPG) production in osteoblast‑like MC3T3‑E1 cells, and that p70 S6 kinase positively regulates the OPG synthesis by BMP‑4. The present study investigated the involvement of HSP90 in the BMP‑4‑stimulated OPG synthesis and the mechanism in MC3T3‑E1 cells. HSP90 inhibitors, 17‑allylamino‑17demethoxy‑geldanamycin (17‑AAG), 17‑dimethylamino‑ethylamino‑17‑demethoxy‑geldanamycin (17‑DMAG) and geldanamycin significantly suppressed the BMP‑4‑stimulated OPG release. Geldanamycin markedly reduced the BMP‑4‑induced mRNA expression of OPG. 17‑AAG and 17‑DMAG significantly attenuated the phosphorylation of p70 S6 kinase induced by BMP‑4 without affecting the BMP‑4‑induced phosphorylation of mothers against decapentaplegic homolog 1/5. The results suggest that HSP90 inhibitors suppress the BMP‑4‑stimulated OPG synthesis in osteoblasts, and that their suppressive effects are exerted through downregulating p70 S6 kinase.

We have previously shown that glycoprotein Ib/IX/V activation stimulates the release of the soluble CD40 ligand (sCD40L) via the generation of thromboxane A2 from human platelets. In the present study, the role of Rac, which is a member of the Rho family, was investigated in the thromboxane A2‑stimulated release of platelet‑derived growth factor (PDGF)‑AB and sCD40L in human platelets. U46619, a thromboxane receptor agonist, stimulated the activation of Rac time‑dependently in human platelets, and NSC23766, a selective inhibitor of the Rac‑guanine nucleotide exchange factor interaction, reduced the U46619‑induced platelet aggregation. NSC23766 markedly suppressed the U46619‑induced p38 mitogen-activated protein (MAP) kinase phosphorylation. The thromboxane A2‑induced release of PDGF‑AB and sCD40L was significantly suppressed by NSC23766 in a dose‑dependent manner. In addition, NSC23766 reduced the sCD40L release stimulated by ristocetin, a glycoprotein Ib/IX/V activator. These results indicate that Rac regulates the thromboxane A2‑induced stimulation of PDGF‑AB secretion and sCD40L release via the p38 MAP kinase in human platelets.

Incretins including glucagon-like peptide-1 (GLP-1) and glucose‑dependent insulinotropic polypeptide (GIP) secreted from the small intestine after oral food ingestion are currently recognized to stimulate insulin secretion from pancreatic β cells. We previously reported that p70 S6 kinase limits the tumor necrosis factor‑α (TNF‑α)‑stimulated interleukin-6 (IL‑6) synthesis in osteoblast‑like MC3T3‑E1 cells. In the present study, we investigated the effects of incretins on the TNF‑α‑induced IL‑6 synthesis and the underlying mechanism in MC3T3‑E1 cells. GLP‑1 and GIP significantly upregulated both TNF‑α‑stimulated IL‑6 release and mRNA levels. Wedelolactone, an inhibitor of IκB kinase, amplified the TNF-α-induced IL‑6 release. GLP‑1 significantly attenuated the TNF‑α‑induced phosphorylation of IκB without affecting the phosphorylation of p70 S6 kinase. On the other hand, GLP‑1 markedly induced the phosphorylation of cAMP response element-binding protein (CREB). H‑89, an inhibitor of protein kinase A, significantly suppressed the enhancement by GLP-1 of TNF-α-stimulated IL‑6 release. Dibutyryl cAMP, a permeable analogue of cAMP, which suppressed the TNF-α-induced IκB phosphorylation, amplified the IL‑6 release. These results strongly suggest that incretins upregulate the TNF-α-stimulated IL‑6 synthesis in osteoblasts, and that the amplifying effect of incretin is exerted via reducing the IκB/NF‑κB pathway through the adenylyl cyclase-cAMP system.

We have previously reported that collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase in human platelets is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble cluster of differentiation 40 ligand (sCD40L). Adenosine monophosphate-activated protein kinase (AMPK), which is known to regulate energy homeostasis, has a crucial role as an energy sensor in various eukaryotic cells. The present study investigated the effects of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (AICAR), which is an activator of AMPK, on the collagen-induced activation of human platelets. It was demonstrated that AICAR dose-dependently reduced collagen-stimulated platelet aggregation up to 1.0 µM. Analysis of the size of platelet aggregates demonstrated that AICAR decreased the ratio of large aggregates (50-70 µm), whereas the ratio of small aggregates (9-25 µm) was increased by AICAR administration. AICAR markedly attenuated the phosphorylation levels of p44/p42 MAP kinase and HSP27, which are induced by collagen. Furthermore, AICAR significantly decreased the secretion of PDGF-AB and the collagen-induced release of sCD40L. These results indicated that AICAR-activated AMPK may reduce the secretion of PDGF-AB and the collagen-induced release of sCD40L by inhibiting HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

AMP-activated protein kinase (AMPK), a key enzyme sensing cellular energy metabolism, is currently known to regulate multiple metabolic pathways. Osteoprotegerin plays a pivotal role in the regulation of bone metabolism by inhibiting osteoclast activation. We have previously reported that prostaglandin D2 (PGD2) stimulates the synthesis of osteoprotegerin through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. On the basis of these findings, we herein investigated the implication of AMPK in PGD2-stimulated osteoprotegerin synthesis in these cells. PGD2 induced the phosphorylation of AMPKα (Thr-172) and AMPKβ (Ser-108), and the phosphorylation of acetyl-coenzyme A carboxylase, a direct AMPK substrate. Compound C, an AMPK inhibitor, which suppressed the phosphorylation of acetyl-coenzyme A carboxylase, significantly attenuated both the release and the mRNA levels of osteoprotegerin stimulated by PGD2. The PGD2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK but not p38 MAP kinase were markedly inhibited by compound C. These results strongly suggest that AMPK regulates the PGD2-stimulated osteoprotegerin synthesis at a point upstream of p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Adenosine monophosphate-activated protein kinase (AMPK) is currently recognized to act as a key sensing enzyme in the regulation of cellular energy homeostasis. It has been previously demonstrated that prostaglandin E2 (PGE2) stimulates the synthesis of osteoprotegerin (OPG) through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. In the present study, it was investigated whether AMPK is implicated in the PGE2‑induced OPG synthesis in MC3T3‑E1 cells. PGE2 was observed to induce the phosphorylation of AMPKα (Thr‑172) and AMPKβ (Ser‑108) in a time‑dependent manner. PGE2 additionally induced the phosphorylation of acetyl‑coenzyme A (CoA) carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which attenuated the phosphorylation of acetyl‑CoA carboxylase, significantly suppressed the PGE2‑stimulated OPG release and the mRNA expression level. Compound C failed to affect the PGE2‑stimulated phosphorylation of p38 MAP kinase or p44/p42 MAP kinase. On the contrary, the phosphorylation of SAPK/JNK was markedly attenuated by compound C. The results of the current study suggest that AMPK acts as a positive regulator in PGE2-stimulated OPG synthesis via SAPK/JNK signaling in osteoblasts.

Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.

Our group has previously shown that αB-crystallin (HSPB5), a small heat shock protein, inhibits human platelet aggregation by ristocetin, an activator of glycoprotein Ib/IX/V. In addition, it was demonstrated that glycoprotein Ib/IX/V activation induces soluble CD40 (sCD40) ligand release via thromboxane (TX) A2. In the present study, the effect of αB-crystallin on the ristocetin-induced sCD40 ligand release in human platelets was investigated. The ristocetin-induced release of sCD40 ligand was suppressed by αB-crystallin. In addition, αB-crystallin reduced the ristocetin-stimulated production of 11-dehydro-TX B2, a stable metabolite of TXA2. αB-crystallin did not suppress the platelet aggregation induced by U46619, a TXA2 receptor agonist. αB-crystallin had little effect on the U46619-induced phosphorylation of p38 mitogen-activated protein kinase or sCD40 ligand release. In addition, αB-crystallin failed to reduce the binding of SZ2, a monoclonal antibody against the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that αB-crystallin extracellularly suppresses ristocetin-stimulated release of sCD40 ligand by inhibiting the TXA2 production in human platelets.

Resveratrol is a natural polyphenol found in red grape skins, berries and red wine. Accumulating evidence suggests that resveratrol has various beneficial effects on the human body. In the present study, we investigated the effects of prostaglandin E(2) (PGE(2)) on osteoprotegerin (OPG) synthesis and the effects of resveratrol on OPG synthesis in osteoblast-like MC3T3-E1 cells. PGE(2) significantly stimulated both the release of OPG and the mRNA expression levels of OPG, as shown by OPG assay and real-time RT-PCR, respectively. Resveratrol markedly suppressed the release and the mRNA levels of OPG induced by PGE(2). On the contrary, SRT1720, an activator of sirtuin 1 (SIRT1), hardly affected the PGE(2)-induced release of OPG. PD98059 [a specific inhibitor of the upstream kinase that activates p44/p42 mitogen-activated protein (MAP) kinase], SB203580 (a specific inhibitor of p38 MAP kinase) and SP600125 [a specific inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)], reduced the PGE(2)-induced release of OPG. Resveratrol attenuated the PGE(2)-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK. However, SRT1720 failed to affect the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK induced by PGE(2). These results strongly suggest that resveratrol reduces PGE(2)-stimulated OPG synthesis through the inhibition of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK in osteoblasts, and that these suppressive effects are independent of the activation of SIRT1.

Resveratrol, a natural polyphenol with health-related properties mainly existing in grape skins and red wine, possesses beneficial effects on human being. We have previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction via activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism behind the effect of PGD2 on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the OPG synthesis in MC3T3-E1 cells. PGD2 significantly stimulated both the OPG release and the expression levels of OPG mRNA. Resveratrol and SRT1720, an activator of SIRT1, markedly suppressed the PGD2-induced OPG release and the mRNA levels of OPG. PD98059, a specific MEK inhibitor, SB203580, a specific p38 MAP kinase inhibitor, and SP600125, a specific SAPK/JNK inhibitor suppressed the PGD2-stimulated OPG release. PGD2-induced phosphorylation of p38 MAP kinase and SAPK/JNK was attenuated by resveratrol or SRT1720. However, resveratrol or SRT1720 failed to affect the phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1), a downstream substrate of Rho-kinase and p44/p42 MAP kinase. These results strongly suggest that resveratrol suppresses PGD2-stimulated OPG synthesis through inhibiting p38 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is exerted at the point downstream of Rho-kinase but upstream of p38 MAP kinase or SAPK/JNK.

Aim We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells. Main methods The levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR. Key findings BMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4. Significance These results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.

Though livers from cadavers being transplanted, more patients are waiting the grafting. Because of this shortage of the donation, artificial liver is expected. But at present, artificial livers do not satisfy the patients yet. In this study, we attempted to establish novel hepatoma cell line for better artificial liver. We have ever introduced anti-apoptosis gene into hybridoma cells and this transfection delayed cell death. Because of this prolonging culture, the transfectants produce 2 times more antibodies than wild type. For the longer-term or permanent artificial liver, we introduced bd-2, anti-apoptosis gene, to HepG2 hepatoma cell.